Method of preparing co-carboxylase



Patented Jan. 30, 1940 v 2,188,323

, 2,188,323 METHOD or PREPARING CO-CARBOXYLASE Henry Tauber, Florence, S. 0., assignor to Merck Co. Inc., Rahway, N. J a corporation of New ersey No Drawing. Application June 28, 1938,

Serial No. 216,387

7 Claims. (Cl. 260251) This invention relates to a method of prepar- By repeating the precipitation two more times, ing a co-carboxylase concentrate, and more parmany of the impurities are removed. A physical ticularly to an easy and eificient method of synyield of about 70% of a, synthetic non-hygrothetically preparing such concentrate. scopic co-carboxylase concentrate is obtained,

Co-coboxylase concentrates may be obtained which shows activity similar to the co-carboxylase 5 from natural sources, for example, from yeast, obtained from natural sources.

but such methods of preparation are extremely Example H tedious and costly.

I have discovered that co-carboxylase concen- The proce o Example I-IS Carried 10 tr t s can b synthetically prepared t good substituting extraction with water at ordinary 10 yield and at relatively low cost by reacting vitatemperature for ct o with Water at the min 131 salts, such as the hydrochloride, hydroboiling point- The physical yield 0f -C b Xy bromide, phosphate, sulfate, etc., with a pyroese concentrate is about phosphate, such as sodium pyrophosphate, and Modifications y be made in carrying Out a mineral acid, such as orthophosphoric acid. this Process Without departing from t Sp r t 15 The reaction may be carried out at between about and Scope of this invention, nd I m to be lim- 100-200 0., best results being obtained at a temited only y he. appendin claims. perature Of about 155 C. I im The following examples illustrate methods of The Process comprising reacting n alkalicarrying out my invention but it is to be undermetal pyrophosphate, Phosphoric c a d a vist d that th examples are by way of m tamin B1 salt at a temperature of between about tration and not of limitation. 100 and 2. The process comprising reacting an alkali- Example 1 metal pyrophosphate, phosphoric acid, and a vi- 1 cc, of 85% orthophosphoric acid is heated in tamin B1 salt at a temperature of about 155 C. a pyrex tube until a slight precipitate forms on 3. The process comprising reacting sodium the side of the tube and dense. fumes appear, pyrophosphate, orthophosphoric acid, and a viand 500 mg. of sodium pyrophosphate, free of tamin B1 salt at a temperature of about 155 C, water of crystallization, are added thereto. 500 4. The process comprising reacting sodium mg. of vitamin B1 hydrochloride are then pyrophosphate, orthophosphoric acid and a vi- 30 added and the mixture heated to 155 C. on an tamin B1 hydrohalide at a temperature of about oil bath for about 3 to 15 minutes, with stirring. 155 C.

The mass is allowed to cool, and 10 cc. of water 5. The process comprising reacting sodium hyare added. A saturated barium hydroxide solurophosphate, orthophosphoric acid and vitamin '3 tion is added untilno more precipitate forms. B1 hydrochloride at a temperature of about 1 The precipitate is centrifuged ofi and shaken 155 C. for five minutes with 40 cc. of water. It is again 6. The process comprising reacting sodium pycentrifuged, 30 cc. of water is added to the preciprophosphate, orthophosphoric acid and vitamin itate, the mixture is boiled for one minute, and B1 hydrobromide at a temperature of about again centrifuged. This procedure is repeated 155 C.

." two more times. The four extracts are added. 7. The process comprising reacting sodium py- 49 together and made slightly acid to congo by the rophosp a p e p e ie cid and vitamin addition of dilute sulfuric acid. The mass is B1 hydrochloride at a temperature of about 155 allowed to stand in the refrigerator for a few 0., dissolving the reaction mass in water, adding hours and the barium sulphate is centrifuged off. barium hydroxide solution to the aqueous solu- 5 cc. of N hydrochloric acid-are added to the tion, re the barium precipitate formed,

solution, and the mixture is concentrated in rep te y Washing the Said precipitate with wavacuo to 30 cc. at about 25 C. At this point a ter, making the combined extracts slightly acid small amount of crystals appear. The mixture is with dilute sulfuric acid, removing the barium cooled in ice water and 10 volumes of a mixture sulfate formed, treating the solution with a mixof two parts ether and one part absolute alcohol ture of absolute alcohol and ether to obtain a are added. Crude co-carboxylase immediately co-carboxylase concentrate. precipitates. It is dissolved in 3 cc. of 0.3 N hydrochloric acid and again precipitated as above. HENRY TAU'BER. 

